Abstract 5560: Connexin 43-Mediated ATP Release Reduces Reperfusion-Induced Barrier Failure in Endothelial Cells
Background: Reperfusion injury impairs endothelial barrier function leading to edema formation impeding recovery of the reperfused heart. Recently we have shown, that ATP either exogenously applied or spontaneously released from coronary endothelial cells (EC) during reperfusion, protects the endothelial barrier against reperfusion injury. Here we tested the hypothesis, that ATP is released via connexin 43 (Cx43) hemichannels and protects against barrier failure during reperfusion.
Methods and results: Cultured EC from porcine aorta were transfected with Cx43-specific or non-specific siRNA 24hrs before onset of ischemia/reperfusion (40min Po2<5mmHg; pH 6.4/40 min, Po2=140 mmHg, pH7.4). Control cells were exposed to normoxia only. ATP release (luciferin-luciferase assay), Cx43 content (western blot), and barrier function (interendothelial gap formation, video-imaging system) were determined. Cx43 silencing revealed that ATP release was closely correlated to Cx43 content. On average Cx43 was reduced by 65±12% resulting in a 58±10% decrease of ATP release (P<05, n =5, for all further parameters). Cx43 silencing as well as pre-incubation of EC with 25μM 18-αglycyrrhetinic acid (GA), a pan-specific connexin inhibitor, reduced the reperfusion-induced ATP release significantly and aggravated endothelial barrier failure by increasing intercellular gap formation from 314±25% to 950±28%. However, increase of intercellular gaps by both maneuvers could be blunted to 370±31% when exogenous ATP (10μM) was added at the onset of reperfusion, indicating that Cx43 silencing did not effect ATP-induced signaling leading to stabilization of EC barrier.
Conclusions: Cx43 hemichannels protect endothelial cells against severe barrier failure during reperfusion. This effect is due to Cx43-mediated release of ATP and its established protective effect on intercellular gap formation by an autocrine mechanism.